High-resolution Raman spectroscopy reveals compositional differences between pigmented incisor enamel and unpigmented molar enamel in Rattus norvegicus

Dental enamel is a peculiar biological tissue devoid of any self-renewal capacity as opposed to bone. Thus, a thorough understanding of enamel composition is essential to develop novel strategies for dental enamel repair. While the mineral found in bone and dental enamel is generally viewed as the biologically-produced equivalent of hydroxy(l)apatite, the formation of these bioapatites is controlled by different organic matrix frameworks—mainly type-I collagen in bone and amelogenin in enamel. In lower vertebrates, such as rodents, two distinct types of enamel are produced. Iron-containing pigmented enamel protects the continuously growing incisor teeth while magnesium-rich unpigmented enamel covers the molar teeth. Using high-resolution Raman spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy, this work explores the differences in acid phosphate (HPO42−), carbonate (CO32−), hydroxyl (OH−), iron, and magnesium content of pigmented incisor enamel and unpigmented molar enamel of Sprague Dawley rats. Bundles of hydroxy(l)apatite nanowires comprise the enamel prisms, where prisms in pigmented enamel are wider and longer than those in unpigmented molars. In contrast to magnesium-rich unpigmented enamel, higher mineral crystallinity, and higher HPO42− and OH− levels are hallmark features of iron-rich pigmented enamel. Furthermore, the apparent absence of iron oxides or oxy(hydroxides) indicates that iron is introduced into the apatite lattice at the expense of calcium, albeit in amounts that do not alter the Raman signatures of the PO43− internal modes. Compositional idiosyncrasies of iron-rich pigmented and nominally iron-free unpigmented enamel offer new insights into enamel biomineralisation supporting the notion that, in rodents, ameloblast function differs significantly between the incisors and the molars.

www.nature.com/scientificreports/ HPO 4 2− being introduced under acidic conditions 12 . HPO 4 2− containing phases such as octacalcium phosphate are frequently encountered in the mineralised dental biofilm 13 . And although human and bovine enamel are believed to contain about 5 wt% HPO 4 2−14 , Raman studies of human premolar teeth have not been able to detect non-apatitic environments 15,16 .
Chemical and structural characterisation of biological tissues using Raman spectroscopy is often plagued by the intrinsic autofluorescence 17 . This process originates from various organic moieties 18 , but can be suppressed by methods such as deproteinisation with sodium hypochlorite (NaOCl) 19 . This work uses high-resolution Raman spectroscopy together with scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) to probe the major compositional differences (particularly the HPO 4 2− , CO 3 2− , and OH − environments) between two distinct types of enamel-pigmented incisor enamel (PIE) and unpigmented molar enamel (UME) in the rat (Rattus norvegicus), with and without deproteinisation. Furthermore, the Raman spectra are compared with geologic hydroxy(l)apatites from the RRUFF™ database 20 , such as the well-characterised and highly crystalline Holly Springs hydroxy(l)apatite 21,22 , which are typical reference materials in crystallographic studies of biogenic calcium phosphates 23 .

Results
The major X-ray emission lines Ca Kα (3.692 keV), Ca Kβ (4.013 keV), P Kα (2.014 keV), Fe Kα (6.403 keV), Fe Kβ (7.058 keV), Fe Lα (0.705 keV), and Mg Kα (1.254 keV) confirmed the differences in Ca/P ratio, Fe content, and Mg content between PIE and UME. The Ca/P ratio of UME (1.30 ± 0.02 at.%) is higher (p < 0.05) than PIE (1.19 ± 0.01 at.%). Similarly, the Mg/Ca ratio of UME (0.011 ± 0.001 at.%) is higher (p < 0.05) than PIE (0.002 ± 0.001 at.%). Whereas the Fe content (Fe/Ca ratio) of UME is negligible, PIE shows significant Fe enrichment (~ 0.15 ± 0.01 at.%), which results in higher (Ca + Mg + Fe)/P ratio (~ 4.2 ± 0.9 at.%), indicating the incorporation of iron at the expense of calcium (Fig. 1). Small amounts of Cl are also detected, which may occur by partial substitution of OH −24 . Scanning electron microscopy (SEM) of the enamel surface reveals that UME is smooth while PIE is grainy in comparison. UME etches homogeneously with orthophosphoric acid (H 3 PO 4 ) but PIE is mildly resistant to acid attack, as evident from the islands of an incompletely removed surface layer. When visualised after acid etching, bundles of hydroxy(l)apatite nanowires comprise the enamel prisms, where PIE prisms are wider and longer than UME prisms.
Raman spectra of enamel and bone were compared to three geologic hydroxy(l)apatites. Remarkable similarities are noted between enamel (PIE and UME) and hydroxy(l)apatite from the Wessels mine (Northern Cape Province, South Africa. RRUFF: R130713) (Fig. 6). An unidentified feature is seen at ~ 853 cm −1 for R130713,

Discussion
Better understanding of enamel composition is essential to develop biomimetic and bioinspired strategies for enamel repair 30 . Some of the recent and highly divergent approaches to repair enamel include protein order/ disorder-guided hierarchical mineralised structures 31 and epitaxially-grown hydroxy(l)apatite crystals 32 . In mineralised biological systems, the presence of iron is often associated with high strength-a prime example being the teeth of the common limpet, thought to be the strongest known biomaterial, where iron-containing filamentous crystals of Goethetite [α-FeO(OH)] comprise the reinforcing phase 33 . Likewise, the presence iron contributes to the overall mechanical properties of rodent pigmented enamel 8 . In the northern short-tailed shrew (Blarina brevicauda), iron pigmentation is not confined to the incisors but exists as a general feature of high stress areas on most teeth 34 . Curiously, iron pigmentation of the dental enamel has also been observed in mammalian species as early as the late Cretaceous period 35 .
In rodents, differences in enamel architecture between unpigmented molar enamel, which forms during embryogenesis, and pigmented incisor enamel, which forms during post-natal life, relate to genetic control of ameloblast differentiation involving distinct mechanisms at these distinct phases of life 36 . Autophagy related 7 (ATG7) protein is essential for the secretion of iron from ameloblasts 37 . Moreover, iron deficiency leads to gross loss of pigmentation and enamel hypoplasia/aplasia 38 . Although amelogenin plays a fundamental role in achieving the precise crystal habit, the enzyme matrix metalloproteinase-20 prevents protein occlusion inside apatite crystals 39 .
Fe enrichment of pigmented enamel is possible through partial substitution of Ca 2+ without major changes in PO 4 3− internal modes 40 , although a constriction in lattice parameters is expected 41 . PIE appears to resist acid attack, which has earlier been attributed to the presence of Ca 2+ and Mg 2+ substituted ferrihydrite 8 . However, in the present work, micro-Raman spectroscopy has not revealed evidence of iron oxides or oxy(hydroxides) in PIE 42 . And though it not straightforward to ascertain the oxidation state of Fe (Fe 2+ or Fe 3+ ) from EDX, alone, Fe-L 2,3 electron energy-loss near-edge structure (ELNES) of pigmented Fe-rich enamel from the rodent Myocaster coypus suggests a predominantly Fe 3+ state 43 . Under the assumption that Fe occupies Ca sites in iron-pigmented enamel, the Fe/Ca ratio of 0.15 equates to ~ 13% Ca substitution and therefore ~ 5.15% mass difference. Ab initio calculations of 42 Ca isotopic substitution for 40 Ca, which equates to ~ 5% mass difference at the Ca sites, have revealed that the expected Raman shifts for vibrational modes above ~ 600 cm −1 (for example the ν 1 PO 4 3− band) do not exceed ~ 1 cm −144 . Here, high-resolution Raman spectroscopy reveals this very small shift in ν 1 PO 4 3− peak position for the first time. In unpigmented enamel, Mg 2+ accumulates within intergranular regions of amorphous calcium phosphate 6,45 . Compared to rat molars, as reported here, the Mg content at the surface of human molars is nearly twice as much at the enamel surface and progressively increases towards the dentinoenamel junction 46 .
Fourier transform infrared spectroscopy studies have suggested the presence of non-apatitic environments (e.g., HPO 4 2− groups) in porcine enamel 47 . Here, high-resolution Raman spectroscopy confirms the presence of HPO 4 2− in both pigmented and unpigmented rat enamel. HPO 4 2− is thought to be a precursor phosphate source for enamel apatite 48 . Therefore, detection of higher HPO 4 2− at the surface of PIE (vs. UME) may be a function of tissue age, as has been reported across different developmental stages of porcine enamel 49 . It has been suggested that acidic conditions favour the fast growth of highly crystalline hydroxy(l)apatite by dissociating calcium phosphate aggregates into Ca 2+ and PO 4 3− ions, which would otherwise block crystal growth and lead to lower crystallinity 50 . If the higher crystallinity and greater HPO 4 2− content of PIE (vs. UME) can be explained by a more acidic environment, it must be determined how this acidic pH is regulated, e.g., if it is biologically driven. Removal of OH − from the local environment through incorporation into the apatite lattice, also more abundant in PIE than in UME, further points towards the presence of acidic conditions. Nevertheless, the OH − content of www.nature.com/scientificreports/ PIE is lower than values of human and boar enamel reported by Pasteris and co-workers 27 . The anticorrelation between CO 3 2− content and crystallinity with little apparent influence of HPO 4 2− warrants further investigation and raises the question whether crystallinity correlates with CO 3 2− only. Organic contamination of UME to a greater extent than PIE is hardly surprising since the latter is continually lost to wear and replaced by pristine mineral. Change in the 428/450 cm −1 ratio of UME, from ~ 1 (indicating high symmetry of PO 4 3− groups) to 0.85 after deproteinisation, suggests a reduction in symmetry and that UME is more susceptible than PIE to alterations. The detection of Ca(OH) 2 points towards the presence of CaO, which readily reacts with atmospheric humidity 51 . Finally, simultaneous increases in mineral crystallinity and CO 3 2− content of bone upon deproteinisation are artefactual and imply loss of recently deposited extracellular matrix and poorly crystalline mineral at the bone surface 52 .
In summary, the chemical contrasts between pigmented and unpigmented enamel in rodents, including HPO 4 2− content, CO 3 2− content, mineral crystallinity, reflect ameloblast function and point towards putative differences in the specific local environmental conditions (e.g., the interplay between pH and the HCO 3 buffer system 53 ) of the organic extracellular matrix and matrix metalloproteinase-20 activity during enamel biomineralisation. While the precise functional role of iron in tooth development remains unclear, iron accumulation in rodent incisors (and the presence of iron in mature ameloblasts) is related to the continuously erupting nature of this tooth 54 . This characteristic feature of rodent incisors also serves to explain the higher HPO 4 2− content of PIE (vs. UME). On the other hand, the high CO 3 2− content of UME is attributed to B-type substitution (i.e., CO 3 2− for PO 4 3− ) typical of biological apatites 55 , and contributes to lower crystallinity together with Mg 2+56 .

Materials and methods
Hemi-mandibles of adult Sprague Dawley rats, obtained as part of an unrelated study, were fixed in 10% neutral buffered formalin, defatted in acetone (~ 30 min), and stored in Hank's Balanced Salt Solution (Gibco™) at 4 °C (Fig. 7). The organic constituents were removed by exposure to 10% NaOCl (3 h at room temperature Micro-Raman spectroscopy. Micro-Raman spectroscopy was performed using a confocal Raman microscope (Renishaw inVia Qontor) equipped with a 633 nm laser and LiveTrack™ focus-tracking technology 57 .
The laser was focussed down on to PIE (labial aspect), UME (buccal aspect), and bone (mandibular ramus) surface using a × 50 objective. The Raman scattered light was collected using a Peltier cooled CCD deep depletion NIR enhanced detector. Using the 2400 g mm −1 grating (348-1104 cm −1 wavenumber range, step size of 0.75 ± 0.04 cm −1 ), Raman spectra were obtained from enamel (9 spots per sample) at 8 s (NaOCl−) or 4 s (NaOCl+) integration time and 10 accumulations, and from bone (one spot per sample) at 10 s (NaOCl−) or 5 s (NaOCl+) integration time and 20 accumulations. Using the 1800 g mm −1 grating (800-3700 cm −1 wavenumber range, SynchroScan wide-range scanning mode; step size of 1.0 ± 0.15 cm −1 ), Raman spectra were obtained from enamel (3 spots per sample, NaOCl+) at ~ 60 s integration time and 10 accumulations. The laser power at the sample was ~ 15 mW. Background subtraction and cosmic ray removal were performed using intelligent spline fitting in Renishaw WiRE 5.4 software.
Statistical analysis. The Wilcoxon signed-rank test was used for statistical analysis. Mean values ± standard deviations are presented and p values < 0.05 were considered statistically significant.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.